Evaluation of the antioxidant effects of melatonin on the larynx mucosa of rats exposed to environmental tobacco smoke.

OBJECTIVES: This study's aim was to investigate the effect of melatonin in terms of mitigating the effects of smoking on the laryngeal mucosa of rats exposed to environmental tobacco smoke. DESIGN: Rats were divided into four groups: Melatonin + Smoking group exposed to smoke with melatonin; Smoking group exposed to smoke without melatonin; Saline group not exposed to smoke without melatonin; Melatonin group not exposed to smoke with melatonin. CuZn-superoxide dismutase (CuZn-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities were evaluated in plasma and tissues. Tissues were also examined the changes of squamous hyperplasia, keratosis, parakeratosis and epithelial hyperplasia by light microscope and the ultrastructural changes by electron microscope. RESULTS: Tissue SOD, CAT and GSH-Px activities were significantly higher in Saline and Melatonin groups than Melatonin + Smoking and Smoking groups. Plasma CuZn-SOD and CAT activities were significantly higher in Saline and Melatonin groups than Smoking group. Plasma GSH-Px showed no significant difference. The rate of epithelial hyperplasia was significantly higher in Smoking group than the other groups. The rate of parakeratosis was significantly higher in Smoking group than the other groups. The epithelial cells in Melatonin + Smoking group displayed, normal cell structure similar to those in Saline group under electron microscope. CONCLUSIONS: The study shows that smoking induces substantial pathological changes in the laryngeal mucosa and melatonin may have some beneficial effects in partially reversing smoking-induced laryngeal injury by inducing the expression of antioxidants; biochemical and histological outcomes also support these findings due to preventing tissue damage in laryngeal mucosa exposed to smoke.

[Expression and significance of DNA-dependent protein kinase in human laryngeal squamous cell carcinoma].

OBJECTIVE: To study the expression of DNA-dependent protein kinase (DNA-PK) in human laryngeal squamous cell carcinoma (LSCC) and normal laryngeal mucosa (NLM), and to analysize the relationship between the expression and the clinicopathologic parameters of LSCC. METHOD: Immunohistochemical technique (Envision) was used to detect the expression of DNA-PK in 64 cases of LSCC and 15 cases of NLM. To investigate an investigation was conducted on the relationship between the expression and clinico-pathological features of LSCC. RESULT: DNA-PK was lowly expressed in NLM and highly expressed in LSCC,the positive rate of DNA-PK expression was 26.67% (4/15), 78.13% (50/64), respectively, and there was significant different difference between the two groups (P < 0.05). Its expression was correlated with the level of histodifferentiation (P < 0.05), but not with TNM stages and neck lymph node metastasis (P > 0.05). CONCLUSION: DNA-PK may be involved in disease development of LSCC.

Differentiated expression of membrane type metalloproteinases (MMP-14, MMP-15) and pro-MMP2 in laryngeal squamous cell carcinoma. A novel mechanism.

Cancer progression involves multiple proteolytic interactions, with metalloproteinases (MMPs) performing a crucial role. MMP-2, a major MMP, plays a key role in the degradation of basement membranes. Mechanisms underlying MMP-2 activation had to be investigated. Membrane-type matrix metalloproteinases are not only responsible for the regulation of extracellular matrix remodeling, but also involved in the activation of several inactive MMPs. The aim of this study was to evaluate the expression of pro-MMP2, MMP-14, and MMP-15 in tumor cells and tumor stroma. Immunohistochemical studies were performed on paraffin-embedded tissue sections including laryngeal squamous cell carcinoma (SCC). We found the expression of pro-MMP2 in 58% of cases, MMP-14 in 78%, and MMP-15 in 98% of cases of SCC. In all tumor cases, we revealed a higher expression of pro-MMP2 in tumor stoma than in tumor cells. The expression of MMP-14 and MMP-15 was higher in tumor cells than in the stroma. Moreover, we found a statistically significant difference between the expression of MMP-14 and MMP-15 in the tumor in comparison with the surrounding stroma (P < 0.05). An analysis of expression levels of MT-MMPs by classification trees showed that the probability of metastases was related to decreased expression of MMP-14 and increased expression of MMP-15. Our results may suggest that tumor cells with low MMP-14 expression invade tumor stroma and form metastases. Probably, in such cases, tumor progression is stimulated by MMP-15 in an MMP-14 independent pathway, a novel (alternative) mechanism.

Pepsin and carbonic anhydrase isoenzyme III as diagnostic markers for laryngopharyngeal reflux disease.

OBJECTIVES/HYPOTHESIS: The objective was to investigate the potential use of pepsin and carbonic anhydrase isoenzyme III (CA-III) as diagnostic markers for laryngopharyngeal reflux disease. STUDY DESIGN: Prospective cell biological investigation was conducted of laryngeal biopsy specimens taken from 9 patients with laryngopharyngeal reflux disease and 12 normal control subjects using antibodies specific for human pepsin (produced in the authors' laboratory within the Department of Otolaryngology at Wake Forest University Health Sciences, Winston-Salem, NC) and CA-III. METHODS: Laryngeal biopsy specimens were frozen in liquid nitrogen for Western blot analysis and fixed in formalin for pepsin immunohistochemical study. Specimens between two groups (patients with laryngopharyngeal reflux disease and control subjects) were compared for the presence of pepsin. Further analyses investigated the correlation between pepsin, CA-III depletion, and pH testing data. RESULTS: Analysis revealed that the level of pepsin was significantly different between the two groups (P < .001). Secondary analyses demonstrated that presence of pepsin correlated with CA-III depletion in the laryngeal vocal fold and ventricle (P < .001) and with pH testing data in individuals with laryngopharyngeal reflux disease. CONCLUSION: Pepsin was detected in 8 of 9 patients with laryngopharyngeal reflux disease, but not in normal control subjects (0 of 12). The presence of pepsin was associated with CA-III depletion in the laryngeal vocal fold and ventricle. Given the correlation between laryngopharyngeal reflux disease and CA-III depletion, it is highly plausible that CA-III depletion, as a result of pepsin exposure during laryngopharyngeal reflux, predisposes laryngeal mucosa to reflux-related inflammatory damage.

[Evaluation of telomerase activation in laryngeal mucosa adjacent to carcinoma].

OBJECTIVE: To investigate telomerase activity in laryngeal nonmalignant mucosa adjacent to carcinoma and discuss its effect on extension and recurrence of laryngeal carcinoma. METHOD: Telomerase repeat amplification protocol was used and combined with histological examination. RESULT: The positive percentage of telomerase activation was even higher in laryngeal nonmalignant mucosa distant toward surgically negative margin of carcinoma than in laryngeal carcinomas themselves (P<0.05). CONCLUSION: Telomerase activation occurred probably before the morphologic alteration, which may imply expansion of the cells with potential of malignant transformation.

Cell biology of laryngeal epithelial defenses in health and disease: further studies.

This is the second annual report of an international collaborative research group that is examining the cellular impact of laryngopharyngeal reflux (LPR) on laryngeal epithelium. The results of clinical and experimental studies are presented. Carbonic anhydrase (CA), E-cadherin, and MUC gene expression were analyzed in patients with LPR, in controls, and in an in vitro model. In patients with LPR, we found decreased levels of CAIII in vocal fold epithelium and increased levels in posterior commissure epithelium. The experimental studies confirm that laryngeal CAIII is depleted in response to reflux. Also, cell damage does occur well above pH 4.0. In addition, E-cadherin (transmembrane cell surface molecules, which have a key function in epithelial cell adhesion) was not present in 37% of the LPR laryngeal specimens. In conclusion, the laryngeal epithelium lacks defenses comparable to those in esophageal epithelium, and these differences may contribute to the increased susceptibility of laryngeal epithelium to reflux-related injury.

Cell biology of laryngeal epithelial defenses in health and disease: preliminary studies.

Esophageal epithelium has intrinsic antireflux defenses, including carbonic anhydrases (CAs I to IV) that appear to be protective against gastric reflux. This study aimed to investigate the expression and distribution of CA isoenzymes in laryngeal epithelium. Laryngeal biopsy specimens collected from the vocal fold and interarytenoid regions were analyzed by Western blotting and immunofluorescence. Carbonic anhydrases I and II were expressed by the majority of samples analyzed. In contrast, CA III was differentially expressed in the interarytenoid samples and was not detected in any vocal fold samples. The expression of CA III was increased in esophagitis as compared to normal esophageal tissue. Carbonic anhydrase I and III isoenzymes were distributed cytoplasmically in the basal and lower prickle cell layers. The laryngeal epithelium expresses some CA isoenzymes and has the potential to protect itself against laryngopharyngeal reflux. Laryngeal tissue may be more sensitive to injury due to reflux damage than the esophageal mucosa because of different responses of CA isoenzymes.

Telomerase activity is upregulated in laryngeal squamous cell carcinoma.

OBJECTIVE/HYPOTHESIS: The immortalizing enzyme telomerase has been linked to carcinogenesis and is being targeted as a novel molecular marker. This study investigated telomerase expression in patients with laryngeal squamous cell carcinoma and correlated telomerase activity with conventional prognostic parameters. STUDY DESIGN: A consecutive series of patients with laryngeal squamous cell carcinoma undergoing surgical salvage for persistent or progressive disease after failed radiation therapy. METHODS: Twenty patient samples of laryngeal squamous cell carcinoma and 20 adjacent histologically normal mucosal samples were assayed using the telomeric repeat amplification protocol (TRAP) method for detection of telomerase activity. The leukemic cell line, K562, acted as a positive control and the human fibroblast line, Hs21Fs, as a negative control. A sample was classified as telomerase positive when an RNase-sensitive hexameric repeat ladder was observed. Absence of laddering was considered a negative result. RESULTS: Seventeen of 20 (85%) tumor samples and 4 of 20 (20%) adjacent histologically normal samples were telomerase positive. No statistically significant difference was observed when densitometric readings were compared by T category, tumor grade, or site (by ANOVA). CONCLUSIONS: Although telomerase activity is present in laryngeal cancer, levels of activation do not correlate with conventional parameters used for prognostication. Our study indicates that the marker may be a useful adjunctive method in the diagnosis of malignancy after radiation failure.

Distribution of NADPH-diaphorase activity in the feline laryngeal mucosa.

We studied nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity in the feline laryngeal mucosa using a histochemical technique in an effort to clarify the role of nitric oxide (NO) in the larynx. Many NADPH-diaphorase-positive nerve fibres were distributed around the blood vessels and the laryngeal glands. The majority of neuronal cells in the intralaryngeal ganglia were NADPH-diaphorase-positive. It is likely that NADPH-diaphorase-positive nerve fibres around the blood vessels and glands in the laryngeal mucosa originate from the intralaryngeal ganglia, and that NO regulates circulation and secretion in the larynx.

Telomerase activity in human laryngeal squamous cell carcinomas.

The activation of telomerase has been shown to be an important step during tumorigenesis in a variety of malignancies and is associated with characteristics of cellular immortality, such as indefinite proliferative potential. We studied telomerase activity in a series of human laryngeal carcinomas. Thirty-six tumors from 35 patients were studied using a sensitive PCR-based technique, the telomeric repeat amplification protocol assay. Telomerase activity was present in 32 tumors (89%), and the level of activity correlated with the stage of disease. In two of four telomerase-negative tumors, we found evidence of an inhibitor of telomerase activity. In many cases, samples of mucosa surrounding the tumor were also studied, and telomerase could be detected in 16 of 21 patients. For this reason, we proceeded to perform a topographical analysis that demonstrated a pattern of telomerase activity suggestive of a spread of telomerase-positive cells. In conclusion, these data indicate that telomerase activation is important for laryngeal carcinogenesis and that telomerase assay might be a valuable addition to determine the spread of the disease.

Up-regulation of urokinase-type plasminogen activator in squamous cell carcinoma of human larynx.

The expression of urokinase-type plasminogen activator (uPA) was investigated in squamous cell carcinoma of the human larynx. For this purpose, tissue extracts from 25 matched samples of normal mucosa and neoplastic larynx were compared for the levels of uPA activity as evaluated by a chromogenic PA assay and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography. Also, uPA antigen was quantified by enzyme-linked immunosorbent assay (ELISA) in 19 cases. The results demonstrate a significant increase in the levels of uPA activity and protein in tumour tissue extracts, more pronounced in tumours with lymph node metastases. Immunohistochemistry performed on 70 biopsies showed that uPA positivity is present both in neoplastic cells and in fibroblast-like cells and macrophages. However, depending on the histological grading and invasive capacity of the tumour, a pronounced intra- and intertumoral heterogeneity in uPA staining was observed. In situ hybridisation confirmed the presence of uPA mRNA in both tumour and stromal cells. The present study provides experimental evidence for a role of uPA in the invasive growth of human laryngeal carcinoma.

Thymidylate synthase activity, folates, and glutathione system in head and neck carcinoma and adjacent tissues.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCC) present variable aggressiveness and chemosensitivity. Because the glutathione (GSH) system and thymidylate synthase (TS) are involved in the resistance to the main drugs used in HNSCC (cisplatin and 5-FU), we studied these systems in tumors and normal mucosae. METHODS: Tumor samples and normal adjacent mucosae were collected from 37 untreated HNSCC patients. GSH and glutathione S-transferase (GST) activity were assayed by spectrophotometry, whereas TS activity and folates were determined by radioassays. RESULTS: Mean GSH levels were higher in tumors (15.2 +/- 8.2 nmol/mg protein) than in mucosae (8.3 +/- 4.1 nmol/mg protein) (p = 0.005, paired t test). GST activity was also higher in tumors (394 +/- 194 nmol/min/mg protein) than in mucosae (261 +/- 132 nmol/min/mg protein) (p = 0.0003). TS activity was markedly higher in tumors (9.2 +/- 21.5 pmol/min/mg protein) compared to that of mucosae (0.9 +/- 1.2 pmol/min/mg protein) (p = 0.0001). Folate levels in tumors and mucosae were similar (1.2 +/- 1.1 and 0.8 +/- 0.9 pmol/mg protein, respectively; p = 0.1, NS). In relation to clinical stage and tumor size, a statistical difference was found in GSH and GST values between tumors and mucosae for stage IV and T3/T4. The increase in tumor TS compared to that of mucosae was significant for all clinical stages, tumor sizes, and nodal involvement. CONCLUSIONS: These data enhance our understanding of the enzymatic systems involved in cisplatin and 5-fluorouracil (5-FU) resistance in HNSCC and normal mucosae and may help to elucidate tumor behavior and interpatient differences in drug sensitivity.

Protein tyrosine kinase activity in laryngeal squamous cell carcinoma.

Oncogenes play an important role in the process of malignant transformation. Since many of the protein tyrosine kinases (PTK) are products of oncogenes, the aim of this study was to demonstrate whether an increased PTK activity could be found in head and neck tumors. By using a non-radioactive dot-blot assay, PTK activity was measured in tumor and normal tissues of 38 patients with laryngeal cancer. The control group consisted of 19 healthy persons. PTK activity in tumor cells was significantly higher (P < 0.001) than in normal cells of the tumor patients and normal controls. Additionally, the PTK activity in the normal mucosa of the tumor patients was significantly higher than in the normal mucosa of the control group.

Effect of beta-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk.

The interaction of bovine beta-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine beta-lactoglobulin displays only one high affinity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 x 10(6) and 2.3 x 10(6) M-1, respectively. However, other binding sites with low affinity are also present. The existence of one high affinity binding site is in accordance with the amount of fatty acids naturally bound to beta-lactoglobulin isolated from milk. The effect of beta-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of beta-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that beta-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.

Lactate dehydrogenase activity in laryngeal carcinoma and surrounding mucosa.

The total lactate dehydrogenase (LDH) activity and LDH isoenzyme pattern were measured in tissue taken from laryngeal cancers and from the laryngeal mucosa both close to and distant from the tumour margin. An increase in the total LDH activity and a cathodic shift in the isoenzyme pattern were found in tumour tissue compared to the close and distant mucosa. However, significant LDH alterations in the mucosa close to the tumour were shown in comparison to the distant mucosa and to the laryngeal mucosa of controls. Histochemical studies showed the LDH activity to be located in the basal and parabasal layers of the epithelium and in the cancer foci.

Urbach-Wiethe disease in O.R.L. practice. (A clinical and histochemical study of the laryngeal lesions).

Urbach-Wiethe disease is a rare disorder, inherited in an autosomal recessive fashion. In this work we have studied the clinical data in six patients with well-established UWD. The histopathological and histochemical findings are discussed. Unless the otolaryngologist has a knowledge of the disease, the diagnosis can be missed.